title: Glycomic analysis by glycoprotein immobilization for glycan extraction and liquid chromatography on microfluidic chip.
abstract: Glycosylation is one of the most common protein modifications and profoundly regulates many biological processes. Aberrant glycosylation is reported to associate with diseases such as cancers, human immunodeficiency virus, and immune disorders. It is considerably important to study protein glycosylation and the associated glycans for diagnostics and disease prognostics. Unlike other protein modifications, glycans attached to proteins are enormously complex. Therefore, the comprehensive analysis of glycans from biological or clinical samples is an unmet technical challenge. Development of the high-throughput method will facilitate the glycomics analysis. In this study, we developed a novel method for the high-throughput analysis of N-glycans from glycoproteins using glycoprotein immobilization for glycan extraction (GIG) coupled with liquid chromatography (LC) in an integrated microfluidic platform (chipLC). The separated glycans were then analyzed by mass spectrometry. Briefly, proteins were first immobilized on a solid support. Glycans on immobilized glycoproteins were modified on solid phase to increase the detection and structure analysis. N-Glycans were then enzymatically released and subsequentially separated by porous graphitized carbon particles packed in the same device. By applying the GIG-chipLC for glycomic analysis of human sera, we identified N-glycans with 148 distinct N-glycan masses. The platform was used to analyze N-glycans from mouse heart tissue and serum. The extracted N-glycans from tissues indicated that unique unsialylated N-glycans were detected in tissues that were missing from the proximal or distal serum, whereas common N-glycans from tissues and serum have mature and sialylated structures. The GIG-chipLC provides a simple and robust platform for glycomic analysis of complex biological and clinical samples.
authors.affiliation: Department of Pathology, Johns Hopkins University , Baltimore, Maryland 21231, United States.
authors.forename: Shuang
authors.initials: S
authors.lastname: Yang
authors.forename: Shadi
authors.initials: S
authors.lastname: Toghi Eshghi
authors.forename: Hanching
authors.initials: H
authors.lastname: Chiu
authors.forename: Don L
authors.initials: DL
authors.lastname: DeVoe
authors.forename: Hui
authors.initials: H
authors.lastname: Zhang
mesh_terms: D000818:Animals; D002853:Chromatography, Liquid / Q000379:methods*; D054794:Glycomics; D006023:Glycoproteins / Q000737:chemistry*; D006801:Humans; D051379:Mice; D044085:Microfluidics / Q000295:instrumentation*; D011134:Polysaccharides / Q000097:blood / Q000302:isolation & purification*; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization
chemical_list: D006023:Glycoproteins; D011134:Polysaccharides